Actinobacillus actinomycetemcomitans is a Gram-negative, facultative anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis (LAP) and with cases of adult periodontitis. This pathogen is the causative agent for other serious infections including infectious endocarditis, soft tissue abscesses, pneumonia, and may contribute to cardiovascular disease. The periodontium is believed to be the source for these non-oral diseases, but little is known about the tropism used by A. actinomycetemcomitans to colonize the oral cavity and to infiltrate and disseminate in tissues. Pathogens have developed diverse strategies to be successful in colonization of host tissues. A common theme amongst these pathogens is the ability to initiate infection by adhesion to specific host macromolecules under stringent or hostile conditions. These molecules include proteins secreted by host cells that form the extracellular matrix (ECM). A. actinomycetemcomitans is found in the connective tissue of the periodontium and in close association with collagen fibers in infected tissues. The bacterium also binds to the ECM proteins, collagen, fibronectin and laminin. Using a genetic approach, we have identified the first A. actinomycetemcomitans collagen adhesin, Ema (extracellular matrix protein adhesin) A and multiple genes involved in regulating the expression of ECM protein adhesin activity. EmaA is structurally related to YadA, a multipurpose ECM protein adhesin of the enteropathogenic bacterium Yersinia enterocolitica, and is associated with bacterial cell surface appendages. These EmaA structures are proposed to be fundamental for collagen adhesion. To elucidate the role of EmaA in colonization and pathogenicity of the bacterium, we propose to 1) map the functional domains of EmaA by determining the collagen binding, subcellular localization, and assembly of surface structures in A. actinomycetemcomitans, 2) investigate the surface structures associated with EmaA by transmission electron microscopy and the role of these structures in resistance to the innate immune response of the host, and 3) determine the number of EmaA molecules required for the assembly of structures on the surface of A. actinomycetemcomitans. A long term goal of the proposed research is to identify and characterize bacterial adhesins that are required for the colonization of the oral cavity and non-oral tissues. These adhesins may serve as targets for future drug development involving small molecules or vaccines that disrupt host-pathogen interactions. Lay statement: Binding to host tissues is the initial phase of all infectious diseases. Understanding how bacteria interact with host cells or tissue constituents will aid in the development of novel therapeutics to prevent initiation or progression of the disease.